Alright.
I'm Erik Jorgensen,
I'm from the University of Utah.
What I'd like to talk about today is
the recycling of synaptic vesicles.
So, before we get started,
I'd like to tell you a bit about synaptic transmission.
So, the way it works is that
there's a cell body that can be very far away
from the synaptic bouton,
an axon that extends
all the way to this synaptic bouton,
and that can be about a meter long.
So, the synaptic bouton
needs to act as an independent organelle.
So, the way this works is that
there are synaptic vesicles
that are filled with neurotransmitter molecules,
and then these molecules,
these synaptic vesicles,
are then docked at the membrane.
And so the way that synaptic transmission works
is that, when there's a calcium influx,
the vesicles then are released,
the vesicle fuses with the plasma membrane,
and releases the neurotransmitter
onto the receptor, shown here.
So, by doing that,
you lose the synaptic vesicle membrane,
and you lose the synaptic vesicle protein.
And so, because that synapse
is so far away from the cell body,
the cell body can't replenish synaptic vesicles,
so what you need to do is recycle them.
And the classic mechanism by which that's done
is clathrin-mediated endocytosis,
and so I'll tell you a little bit about that today.
And then, in the second half,
in the second video,
I'd like to tell you about
some recent work of ours,
demonstrating that there is a second mechanism
for endocytosis at synaptic boutons,
and this is called ultrafast endocytosis.
And in this case, what we see is that
the synaptic vesicle membrane
is recovered very rapidly,
about 30-300 milliseconds,
generating a synaptic endosome,
and then that synaptic endosome
is resolved by clathrin-mediated vesicle budding,
and this is also a very fast process
-- it only takes about 3 seconds.
And then, finally,
when that budded vesicle is uncoated,
we've regenerated a synaptic vesicle
after only 5 seconds,
and so this makes a synapse
be able to run at much faster speeds
than with clathrin-mediated endocytosis.
So, again, we have two videos
that I'll be filming.
The first is this demonstration
of how synapses work.
This is the work of Bernard Katz,
showing that vesicles have this exocytic property.
Secondly, I'll tell you about the classic work
of Heuser and Reese,
demonstrating that vesicles are recycled
via a clathrin-mediated process.
In the second video, I'll tell you about this recent work
identifying this ultrafast pathway.
So, first of all, this work all relied on
the work of Bernard Katz,
who was working in the '50s and '60s,
and this is work that won him the Nobel Prize,
and what he was doing was recording
from the frog sartorius muscle.
So, he had a sharp electrode,
shown here,
penetrating the muscle cell,
and he could then record
changes in the potential of that muscle cell.
And so, what he'd see is that,
as long as there were no changes,
the potential was constant,
at about -60 millivolts (mV).
However, he found that
if he manipulated a pipette
with acetylcholine in it (ACh),
and spritzed on a little acetylcholine onto the muscle,
then he would see a profound depolarization,
shown here.
And what he didn't know at the time,
but we know now,
is that on the membrane of the muscle
are acetylcholine receptors,
so that the acetylcholine molecules,
shown in yellow, here,
bind receptors,
and these receptors are ion channels,
so they open and allow cations
to enter the cell
and cause this depolarization that you see here.
What he then found was that,
as he reduced the amount of
neurotransmitter released onto the muscle,
he saw a proportional decrease in the response,
and he could continue that until
the response was zero.
In other words, there is
no minimal response for a synapse
or for a response to neurotransmitter.
So, this was much different than what he saw
if he left the motor neuron in place.
So, here is a motor axon, shown here...
here's the motor neuron
and here's the motor axon,
and it's now forming a synapse on the muscle cell.
And so, now, he simply records.
He's not spritzing on neurotransmitter,
he's just seeing if there are spontaneous events,
and what he saw are
these spontaneous depolarizations of the muscle.
Moreover, when he looked at them,
they were of a fixed size.
So, from this, he said that
neurotransmission, somewhat like quantum physics,
is a quantal response.
There is a minimal response
that the muscle will respond to.
So, Katz was aware of
electron micrographs that had just recently been done
of the neuromuscular junction
-- this one's by David Robertson --
and, as you can see here,
there are synaptic vesicles
that can be seen in this micrograph.
And so, the way that Katz thought about this was
that maybe these vesicles are filled with neurotransmitter,
their size determines that
there's a fixed amount of neurotransmitter in them,
and that, occasionally,
these spontaneously fuse with the plasma membrane.
And so that's shown, here.
And when that occurs,
a few of these ligand-gated ion channels will be opened,
and so you'll see a fixed response.
So, in this case, it's a depolarization.
Here's we're showing a miniature current,
and so these events are called minis.
And so, the quantal hypothesis
has now become the vesicle hypothesis,
so the idea now is that
the reason that there is a quantal response at synapses
is because of these vesicle fusions
having a fixed amount of neurotransmitter.
And so, these...
I just want to step back for a moment
and tell you a little bit about how these images were created.
They're created by an electron microscope.
First, what you have to do is
embed your sample in plastic.
In this case, I have a worm, a nematode, C. elegans.
It's fixed and then embedded in plastic.
Those plastic sections are then cut,
or the plastic block is then cut into sections.
Those sections are then picked up,
a ribbon of those is picked up onto this grid,
when is mounted on a mount
that will go into the electron microscope,
and then you get an image of the cross-section of the worm,
as shown here.
And so the way the electron microscope works
is that you have an electron source,
the electrons then pass through your sample,
which is on a thin film,
and then the electrons pass through onto a film,
onto photographic film,
and then that generates this image
that you see.
So, let's return, now,
to the quantal hypothesis.
So, Katz understood that
what he needed to demonstrate
is that these synaptic vesicles
fused with the plasma membrane.
And at the time, that was not widely accepted.
People imagined that these synaptic vesicles
could contain neurotransmitter,
but they imagined that the neurotransmitter
was being released directly through the plasma membrane,
not via these synaptic vesicles.
So, to prove the quantal hypothesis,
to prove the vesicle hypothesis,
Katz needed to demonstrate that
these vesicles fused with the membrane,
and that had never been seen before.
And so he took on a grad student,
his name is John Heuser,
and John's job was to try to capture these events.
And he was never able to do so,
and in fact John never got his PhD
working with Katz,
or ever, for that matter.
But he was obsessed with this problem
and wanted to pursue it.
So, he then moved to the lab of Tom Reese,
and together they worked out, I think,
one of the most exquisite experiments
in the history of science,
to prove that these vesicles actually fused.
So, let me tell you a little bit about that.
So, some of those experiments
were done at the Marine Biological Laboratory
in Woods Hole,
and these experiments were done in the 1970s,
and we'll return to the scene of the crime later,
in the second video.
So, here's Tom and John in the 1970s,
when these experiments worked
-- beautiful young men,
dressed appropriately for the time,
so times have changed.
So, what I want to show, here,
is this device that they built.
So, what they did was
they took a piece of the frog sartorius...
pectoral muscle,
which is very thin and could freeze quickly,
and that's why these selected it,
and they left the nerve attached,
so the nerve is still attached, here,
and they wrapped that around a stimulating wire, here.
And this was all mounted on this cylinder,
which was then held in place
by a rod on this device, shown here.
And so, here is where the sample is,
and then if you look up here
you can see this rod that is holding this in place,
and this was held in place by a solenoid,
so just this just a friction connection.
Below, here, is a copper block,
cooled in liquid helium to 4 Kelvin,
so this is the temperature of outer space,
right... at this temperature,
all molecules stop movement.
So, then was they could do is
they could release that solenoid,
and then, using standard physical equations
of acceleration,
calculate the time it would take
for the sample to hit this copper block.
There's a shutter that is removed,
and then there's a trip,
so that at 5 milliseconds before it hits the surface...
so, it's falling, falling, falling, falling...
5 milliseconds before it hits the surface,
that stimulator then activates that nerve,
that nerve then causes neurotransmission to occur,
and vesicles are fusing,
and then... boom.
It hits the copper block and then all movement stops.
So, what did they see?
These beautiful images.
So, here, you can see
an unstimulated neuromuscular junction,
you can see that there are vesicles
that are docked at the membrane,
and when they stimulated and then froze,
5 milliseconds later,
you see that there are these fusions that have occurred.
If you look here,
here's a vesicle that has just fused,
here's another one that's just fused,
and then here's one that's beginning to flatten out,
so it collapses into the plasma membrane.
And so I love this experiment, because it
required knowledge of physics,
it required engineering
to be able to build this device,
and they combined this with a deep understanding of biology,
to say, how are we going to prove
this biological point?
We're going to have to invent
a new instrument.
So, it was really a wonderful experiment.
So, by doing so,
the quantal hypothesis was proved correct.
So, there's a few other
techniques that they applied to this problem,
and one of these is freeze-fracture electron microscopy.
So, in the images I just showed you,
those are very thin sections
that are mounted in a transmission electron microscope.
So, what they also discovered
was if they took that frozen block,
that they could strike it with a knife
and split the ice in half,
and what happens is
the ice will break along the weak points.
So, the weak point
is at that lipid bilayer,
because there's no covalent bonds between that lipid bilayer, right?
They're just interacting with each other
because they're greasy.
So, what happens when you hit that block of ice
is that those split open
and you get two faces of that ice block,
and so on one face is the extracellular layer,
shown here,
and the other is this cytoplasmic layer, shown here.
And so, if you look at those images
independently, if you look at the cytoplasmic layer
that's shown, here,
and if you look at the extracellular layer...
oh, that's the extracellular layer,
excuse me,
and this is the inside, or the cytoplasmic layer,
and you can see these little bumps there,
and those are proteins that are
embedded in the membrane.
So, they can image this because they
spatter it with platinum,
and what that does is provide contrast
that will be visible in an electron microscope,
and it also provides a shadow,
so things look like they're three-dimensional.
And so then they're imaged in a transmission electron microscope,
and that's how you get these beautiful images.
So, now, they're going to take this technique
and they're going to apply it
to stimulated synapses
and see, what do those events look like
in the plasma membrane?
And that's shown here.
So, here, at 0 milliseconds
in an unstimulated synapse,
you see these little bumps,
and these little bumps
are likely to be calcium channels.
And, remember that I told you that
calcium was required for synaptic vesicle fusion.
5 milliseconds after the stimulation,
they see that there are these invaginations,
and what these invaginations represent
is that there's a synaptic vesicle on the other side of this screen,
and it's just fused with the membrane,
and it's created a neck.
And so when you split that,
it breaks across that thin part of the neck,
because you can't cut all the way
into that concave membrane,
and you get these little discs,
and so that is just simply the ice itself,
at that neck.
And so, once again, they demonstrated
that synaptic vesicles fuse with the plasma membrane.
So, at this point, it looked like the fusion took 5 milliseconds,
but we now know from electrophysiological experiments
that that fusion is actually much faster.
After the calcium transient,
after the increase of calcium,
we know that it only takes
one-tenth of a millisecond
for that vesicle to fuse with the plasma membrane.
Okay, so that is the proof that vesicles
contain neurotransmitter
and that neurotransmission
is mediated by these vesicle fusions.
What I'd like to talk about
in the second half of this video
is what happens after that,
what happens to that membrane after it's fused.
So, synapses have to be very robust,
so they can fire...
the neuromuscular function
can fire 100 times a second,
and so every time you do that
you're releasing vesicles.
And there's a limited pool,
maybe 300 synaptic vesicles at a synapse,
so you can see that at these rates
you would deplete the synapse of synaptic vesicles.
And, again, it's far from the cell body,
so the synapse needs to
recycle the synaptic vesicles.
And so, Heuser and Reese realized that,
and so they looked for evidence of that.
And so, the first experiments they did
is they stimulated very intensely
and then they would fix the sample
in a fixative,
and embed them in plastic,
and look at the sections,
to see whether there was any evidence of
recovery or recycling of these synaptic vesicles.
And so that's shown here in this image...
you can see that there are
these invaginations that are occurring,
here's one,
here's another one, here...
and if you look at those, they have these coats on them,
these electron-dense coats,
and those had been seen before,
and this occurs in any cell that is
endocytosing material, like receptors or membrane,
and those are clathrin coats,
and they'd been seen in electron micrographs before.
So, now there was a sort of a unified view
of how you recovered proteins in membranes,
even at synapses,
and it's done via this clathrin-mediated mechanism.
So, this was not...
it didn't show any timing,
this was simply a highly stimulated synapse
that was fixed,
and so the issue is,
how long after stimulation
did these vesicles get recovered?
And so, once again, they turn to the freeze slammer.
So, now, what they do is they have their sample,
and they stimulate,
and they just hold it,
for 1 second, for 3 seconds,
for 10 seconds, for 30 seconds,
and then they let it go
and then they freeze it on this copper block.
So, what did they see?
So, these again are these freeze-fracture experiments,
and at 0 milliseconds --
once again, this is just our control,
it's unstimulate --
you can see that these buds, here,
these buttons, are probably calcium channels.
At 5 milliseconds, you see these fusions
and then nothing happens,
so the synapse is basically quiet.
Until 30 seconds,
you begin to see these invaginations.
And so some of these invaginations
look like proteins are coalescing.
In other images,
you can see that they look deeper,
as if they're being pinched off,
so this suggested that
this was the endocytic event,
and it's not so dramatic here,
but, if they provided a higher stimulation,
then they would see many of these invaginations
occurring at 30 seconds.
So, now we understand...
we have a dogma of how synaptic transmission works.
Vesicles fuse with the plasma membrane,
release neurotransmitter,
the proteins that make up that synaptic vesicle
and the membrane that makes up that synaptic vesicle,
has to be recycled,
and so that's via clathrin-mediated mechanism.
Clathrin forms this superstructure,
a geodesic dome over this piece of membrane,
and then removes it from the surface
to regenerate a vesicle.
This clathrin coat is
then removed to regenerate a vesicle.
So, the two important conclusions
from this video
is that endocytosis, synaptic vesicle recycling,
occurs via a clathrin-mediated mechanism,
and it's rather slow
-- it takes about 30 seconds for these vesicles
to be regenerated.
So, that is the conclusions for this video.
I've told you about how
synaptic vesicles exocytose,
how they fuse with the plasma membrane,
and how we understand
that that is how a synapse works.
And in the second half of this talk,
I told you about how clathrin-mediated endocytosis
recovers that membrane,
recovers those proteins,
to regenerate synaptic vesicles
that can fuse again.
In the second video,
I'd like to tell you about some work...
that there is a parallel system,
that works on a much faster timescale,
to recycle synaptic vesicles.
Thanks once again for listening,
and I encourage you to watch the second video.